The goal of this project is to carry out a large scale screen of small molecular compounds for both antagonists and agonists of human MrgX1 with therapeutic implications of anti-itch and anti-chronic pain, respectively. Primary sensory neurons in dorsal root ganglia (DRG) play essential roles in initiating and mediating pain and itch. We have shown that Mrgs, a family of G protein-coupled receptors specifically expressed in DRG neurons, function as itch receptors as well as endogenous inhibitors of chronic pain. Therefore, modulating Mrg activation offers a unique and promising target to treat chronic pain and itch. We have successfully carried out a pilot screen of ChemBridge Diversity library (20,000 compounds) using a two-stage and three-addition scheme for human MrgX1 agonists, antagonists, and allosteric modulators. The results of the pilot screen are presented in the proposal. In Aim I, we propose to screen a larger and more diverse compound collection (i.e. the MLSMR library) for agonists, antagonists, and allosteric modulators of human MrgX1 with the same protocol as used in the pilot screen. In Aim II, we will confirm positive hits from the primary screen by multiple secondary assays which have been optimized for different purposes of validation screen, confirmatory screen, counter screen, alternative screen, and orthogonal assay. In addition, we will examine the efficacy, potency, and specificity of positive hits using native DRG neurons from wild-type and Mrg-deficient mice monitored by Ca2+ imaging as well as whole-cell patch recordings. Criteria are set for the secondary assays to select potent and specific hits with drug likeness. In Aim III, we will test the potency and specificity of MrgX1 modulators in mouse behavioral assay. We will examine the inhibitory effects of MrgX1 agonists and allosteric agonists on chronic pain as well as the anti- itch effects of antagonists. We have generated, developed, and obtained all reagents necessary for the proposed experiments and have worked out all experimental procedures. In addition, we have great support from our collaborators Johns Hopkins Ion Channel Center and Neurotranslation program at Johns Hopkins University Brain Science Institute on both high throughput screen and medicinal chemistry. These active compounds may be exploited as a therapeutic remedy of chronic pain and itch.